Sample Collection

Endometrial cytology was reported to be a clinically useful procedure in the veterinary literature as early as 1961.³ Since then, many other clinical and controlled investigations have reported on cytology’s usefulness, interpretation, and validity as a diagnostic procedure for broodmares.^4–24

The methods for collection of the sample for endometrial cytologic examination range from dry or moistened sterile or nonsterile cotton or calcium alginate swabs, to loop or scraping devices, to low-volume fluid washing or flushing of the uterine lumen. Calcium alginate swabs are preferred over cotton swabs to avoid cotton fiber shedding on the slide.¹¹ The methodology employed makes little clinical difference as long as (1) the procedure does not harm the endometrium or introduce pathogens to the uterine environment, and (2) the sample collected contains sufficient numbers of endometrial and other cells to enable a reliable cytologic examination. For the practitioner the chosen procedure should be relatively quick and easily performed with common supplies available in the clinic or carried to the farm.

The mare should be prepared with a tail wrap and a perineal scrub using water, cotton, and a mild nondisinfecting soap, then thoroughly rinsed again with clean water and dried with clean paper towels. The practitioner should wear a sterile, shoulder-length, plastic obstetric sleeve, and a sterile, nonbacteriostatic obstetric lubricant should be placed on the back of the gloved hand. The sterile collection device should be guarded by the gloved hand and guided through the vulva, into the vagina, avoiding the urethral opening ventrally, and advanced cranially until the cervix is identified. With the finger(s) the external cervical os should be gently invaded and the full length of one or two fingers inserted through the length of the cervix, stopping at the body of the uterus. The collection device is then carefully guided between the fingers and placed into the uterus.

This basic procedure is used whether a guarded sterile cotton or calcium alginate swab is used to collect samples or whether an artificial insemination or sterile flushing catheter is used for saline wash or lavage to collect the sample. Before the guarded swab is introduced, a thorough vaginal and cervical examination can be performed digitally, thus conserving time and minimizing the number of times the mare’s tract is invaded.

It is possible to recover samples simultaneously for both endometrial culture and cytology using the same sterile, guarded, uterine culture device (Accu-Culshure) without risking contamination of the sample for culture, provided a sterile glass slide is used. Other guarded and nonguarded endometrial culture devices with various designs are available. The key point is to guard the swab with the hand or through the design of the collecting instrument to avoid contamination of the cytologic specimen recovered from vulval, vaginal, and cervical cells. Guarded swab devices can also be used with vaginoscopy as an alternative to the sterile sleeved or manual approach.

An alternate method for collection of endometrial cells is a low-volume fluid wash or lavage. After insertion of the sterile insemination pipette into the uterine body, 50 ml of sterile 0.9% sodium chloride solution is rapidly flushed into the uterus using a 60-ml syringe attached to the distal end of the pipette. The practitioner’s index finger remains inserted through the cervix to guide and manipulate the tip of the pipette as the fluid is immediately withdrawn by negative pressure applied with the attached syringe. Some side-to-side and dorsal-to-ventral movement is required to find the pockets of fluid for aspiration. In most cases, 1 to 5 ml can be recovered. After recovery the pipette tip is guarded again on exit from the mare, and the total aspirate from the syringe and pipette lumen is placed into a clean or sterile tube or other container for processing.

The washing or flushing method of sample recovery for endometrial cytology may provide a more representative sample of the cells contained within the lumen of the uterus compared with the swab technique.²⁰ Although this concern may be justifiable in some mares, the guarded swab technique is used more for practical reasons (eg, simultaneous samples for cytology and culture). An endometrial biopsy sample for histopathology can then be collected after the samples for culture and cytology have been obtained, to avoid contaminating them with blood.

Sample Processing

Precleaned, sterile microscope slides are prepared and sterilized inside plastic or cardboard mailing packets. An additional outer envelope sealed before sterilization helps to ensure long-term sterility during transport and storage. If the sample was obtained from a guarded swab with a cotton or calcium alginate tip, the protective sheath is removed, and the tip of the device is gently rolled across the length of the slide in several motions to displace recovered cells onto the sterile slide surface. It is good practice to make two slides for every cytologic examination to allow the practitioner to stain one for immediate evaluation. The second slide serves as a backup slide in case the other is broken or damaged in transport and as an alternate slide to stain using another method. The tip of the same device is then placed into a sterile microbiologic transport medium for preservation of bacterial pathogens that may have been recovered.

When the Accu-Culshure device is used, the tip of the collecting sheath is wiped with an alcohol-moistened gauze or a clean paper towel, and the material from the tip of the device is pushed onto the sterile slide. Another clean slide is used to drag or smear the collected material across a portion of the surface of the receiving slide. The sample for bacterial culture recovered by Accu-Culshure is separated from the area where the sample for cytologic sample is recovered. The portion for microbial culture is then appropriately processed according to the instructions with the device. The slides prepared in this manner are then placed back into their slide mailer boxes for transport to the practitioner’s office or mailing to a laboratory. The slides can be allowed to air-dry, or they can be immediately stained using Diff-Quik on the farm or in the clinic’s facility.

Samples that are obtained using a saline wash, flush, or lavage technique require more processing than the samples obtained using the guarded swab techniques. The collected fluid sample can be placed into a sterile or clean test tube or placed into a test tube containing 5 ml of 40% ethanol preservative. The fluid samples must be centrifuged and the cytologic smear prepared from the sedimental cells in the bottom of the tubes after centrifugation.

Staining Methods

Air-dried or ethanol-fixed slides can be stained immediately with modified Wright’s or Diff-Quik stain. Air-drying has some trade-offs compared with wet fixation of cytologic specimens. Air-drying preserves cellular material (less loss) but sacrifices cellular detail. Samples that are heavily contaminated by mucus when air-dried will have dark staining of this mucoproteinaceous material that may mask the observation of cellular content on the slide. Wet fixation tends to float some mucoproteins off the slide, preserving cellular detail. When guarded swab recoveries of samples for endometrial cytology have a moderate to heavy mucus contamination, the smeared slides should be immediately stained with Diff-Quik rather than allowing them to air-dry. Alternatively, the mucus-contaminated swab can be placed into a saline or 40% ethanol solution and centrifuged to recover the cellular content of the sample. Diff-Quik provides adequate staining and cellular detail for most routine endometrial cytologic examinations. Alternative stains include rapid Papanicolaou trichrome stain²² for wet-fixed samples and Gram’s-iodine, Wright’s, or Wright’s-Giemsa stain for air-dried samples.